JP2013083490A - Marker for diagnosing gastrointestinal cancer, and method for inspecting gastrointestinal cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a marker for diagnosing gastrointestinal cancer which can be used for an inspection method for simply inspecting gastrointestinal cancer at an early stage, and a method for inspecting gastrointestinal cancer for simply inspecting gastrointestinal cancer at an early stage.SOLUTION: A marker for diagnosing gastrointestinal cancer is used for identifying presence/absence of affection with gastrointestinal cancer, is an N-coupling type sugar chain to be separated from glycoprotein contained in blood and has characteristics in sugar chain arrangement. In the marker, the mass-to-charge ratio of the peak by mass analysis by using a MALDI-TOF-MAS type analyzer is any of 2521/mz, 2216 m/z, 2054 m/z, 2681 m/z, and 3108 m/z.

Description

  The present invention relates to a marker for diagnosis of digestive organ cancer and a method for examining digestive organ cancer.

  The digestive system is an organ responsible for food intake, transport, digestion and nutrient absorption and excretion. Among the digestive organs, the pancreas is an organ with a length of about 15 cm on the back side of the stomach, and the main diseases are pancreatic cancer and pancreatitis. Especially, pancreatic cancer has recently increased in Japanese mortality. It is known as one of the cancers.

  The cause of pancreatic cancer has not been completely elucidated, but it is thought that risk factors include excessive intake of animal fat, protein, alcohol, etc. due to westernization of dietary habits or smoking. Furthermore, people with a history of chronic pancreatitis, pancreatic stone disease, diabetes, and acute pancreatitis are also considered to be a high-risk group for pancreatic cancer.

  Pancreatic cancer is cancer that originates from cells that have an exocrine function, particularly cells of the pancreatic duct through which pancreatic juice flows, and more than 90% of pancreatic cancers are of this type. Pancreatic cancer is extremely high in malignancy, and metastasis to other organs occurs at an early stage, so early detection is an issue.

  However, because the pancreas is surrounded by many organs such as the stomach, duodenum, spleen, small intestine, large intestine, liver, and gallbladder, it exists in the retroperitoneum. It is extremely difficult and is often found in advanced cancer.

  Gastric cancer is a high incidence region in Japan and Southeast Asia, accounting for the second largest cancer mortality rate in the world. Although the prognosis of gastric cancer has improved due to the development of diagnostic techniques and therapies, the prognosis of advanced gastric cancer is still not good, and the 5-year survival rate of gastric cancer that has infiltrated the gastric serosa is as low as 35%. .

  One of the main causes of recurrence after curative resection of advanced cancer is peritoneal dissemination. Although advances in chemotherapy have seen therapeutic effects on recurrence of peritoneal dissemination, the 5-year survival rate is still low. It is known that many steps and many genes are involved in the mechanism of peritoneal dissemination of gastric cancer. It is reported that adhesion molecule-related genes, apoptosis-related genes and other genes are deeply involved in peritoneal dissemination of gastric cancer. Further research is also needed to elucidate the mechanism of metastasis of gastric cancer including peritoneal dissemination of gastric cancer.

  Furthermore, about half of esophageal cancers occur in the middle thoracic esophagus. Over 90% of Japanese esophageal cancers are squamous cell carcinomas. Both morbidity and mortality are higher in men, more than five times that in women. Regarding the carcinogenesis of esophageal cancer, the involvement of drinking and smoking has been reported for a long time, and the involvement of ALDH2 has been reported in recent years. Esophageal cancer surgery is highly invasive and has a poor prognosis.

  In recent years, with the increase of early cancer, cases that can be cured by endoscopic treatment are increasing. In addition, the effects of chemoradiotherapy have been observed, and improvement in treatment results has been observed. However, the prognosis for unresectable advanced cancer remains poor. In the future, establishment of a new screening system for early cancer detection and advancement of chemotherapy are desired.

  By the way, the measurement of a tumor marker is mentioned as a test | inspection / diagnostic method of the cancer developed so far.

  Examples of blood tumor markers for pancreatic cancer diagnosis include CA19-9 (see Non-Patent Document 1), Dupan-2 (see Non-Patent Document 2), and CA-50 (see Non-Patent Document 3). , Span-1 (see Non-Patent Document 4) and the like have already been developed. Also, several patent publications disclose methods for examining or diagnosing pancreatic cancer by using a gene specifically expressed in tumor cells as a marker. To date, PANCIA and PANCIB (see Patent Document 1) and KCCR13L (see Patent Document 2) have been disclosed as pancreatic cancer marker genes. In addition, since there is amplification or deletion of DNA at a specific site of the chromosome of cells of pancreatic cancer, there is also a method for diagnosing pancreatic cancer by detecting the amplification or deletion of the specific chromosome site of the pancreatic cancer. Proposed.

  In addition, gastric cancer is characterized by using a change in the expression level of a known type I collagen C-terminal non-3 heavy chain telopeptide (ICTP) as a marker. The number of demethylated DNA present in DNA repeat sequences obtained from appropriate diagnostic markers for advanced gastric cancer, particularly Skills gastric cancer (see Patent Document 3), and cancerous or non-cancerous tissue specimens, and the ratio Based on the above, a method for determining whether the prognosis of various cancer diseases including gastric cancer is good has been proposed (see, for example, Patent Document 4).

  Furthermore, a method for screening metastatic colorectal cancer or primary or metastatic gastric cancer or esophageal cancer using the expression of SI, CDX1, or CDX2 as an index has been proposed (see Patent Document 5).

  However, tumor markers are currently used to understand the dynamics of advanced malignant tumors, and cancer screening / diagnosis methods using tumor markers that can be used for early diagnosis have been established. Absent.

  In particular, pancreatic cancer is difficult to diagnose at an early stage, and treatment results are poor, so it is urgent to develop a method that can be applied to screening.

Special Table 2000-502902 Japanese Patent Application No. 2003-041843 JP 2001-33460 A JP 2002-112799 A Special table 2003-532389

Somatic Cell Genet., 5, 957-972, 1979 Cancer Res., 42, 601, 1982 Int. Arch. Allergy Appl. Immunol., 71, 178-181, 1983 Nichigai, 87, 236, 1986

  An object of the present invention is to provide a marker for diagnosing digestive organ cancer that can be used in an inspection method for inspecting digestive organ cancer early and simply, and a method for inspecting digestive organ cancer that can be inspected early and easily. There is.

Such an object is achieved by the present invention described in the following (1) to (12).
(1) Used to identify the presence or absence of gastrointestinal cancer,
N-linked sugar chains released from glycoproteins contained in blood,
The N-linked sugar chain is at least one of those represented by the following formula (1) to the following formula (6).

  (2) In the above (1), the N-linked sugar chain represented by the formula (1) has a peak mass to charge ratio of 2521 m / z by mass spectrometry using a MALDI-TOF-MS analyzer. The marker for a digestive organ cancer diagnosis as described.

  (3) In the above (1), the N-linked sugar chain represented by the formula (2) has a peak mass to charge ratio of 2216 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. The marker for a digestive organ cancer diagnosis as described.

  (4) In the above (1), the N-linked sugar chain represented by the formula (3) has a peak mass to charge ratio of 2216 m / z by mass spectrometry using a MALDI-TOF-MS analyzer. The marker for a digestive organ cancer diagnosis as described.

  (5) In the above (1), the N-linked sugar chain represented by the formula (4) has a peak mass-to-charge ratio of 2054 m / z by mass spectrometry using a MALDI-TOF-MS analyzer. The marker for a digestive organ cancer diagnosis as described.

  (6) In the above (1), the N-linked sugar chain represented by the formula (5) has a peak mass-to-charge ratio of 2681 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. The marker for a digestive organ cancer diagnosis as described.

  (7) In the above (1), the N-linked sugar chain represented by the formula (6) has a peak mass to charge ratio of 3108 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. The marker for a digestive organ cancer diagnosis as described.

  (8) The marker for diagnosing digestive organ cancer according to any one of (1) to (7), wherein the digestive organ cancer is pancreatic cancer, esophageal cancer, or stomach cancer.

(9) A test method for digestive cancer that identifies the presence or absence of digestive cancer using the marker for diagnosis of digestive organ cancer according to (1) to (8) above,
A detection step of detecting an N-linked sugar chain represented by the formula (1) to the formula (6) released from the glycoprotein;
A method for examining digestive organ cancer, comprising: an identification step for identifying the presence or absence of the digestive organ cancer based on the detection result of the detection step.

  (10) In the detection step, the N-linked sugar chain represented by the formula (1) to the formula (6) is detected by mass spectrometry using a MALDI-TOF-MS type analyzer (9) The test | inspection method of digestive organ cancer of description.

  (11) When at least one detection value of the mass-to-charge ratio [m / z] is lower than detection values of 2521, 2216, and 2054 is identified as suspected of having digestive organ cancer The method for examining digestive organ cancer according to (10) above.

  (12) When the detection value of at least one of the mass-to-charge ratio [m / z] is higher than the detection value of 2681 and 3108, the disease is identified as suspected of having digestive organ cancer. (10) The method for examining digestive organ cancer according to (10).

  By detecting a marker for diagnosis of digestive organ cancer of the present invention, that is, a specific N-linked sugar chain, from blood collected from a subject, it is possible to easily test for digestive organ cancer at an early stage. It becomes possible.

  Therefore, by the digestive cancer test method using such a marker for digestive cancer diagnosis, it is possible to easily perform digestive cancer test at an early stage of cancer and to treat early digestive cancer. be able to.

It is a graph which shows the result of having quantified the abundance in each sample about the sugar_chain | carbohydrate determined to have a significant difference by T-test performed with respect to 2 groups of a healthy person and pancreatic cancer. (The total signal amount of sugar chain abundance was calculated and corrected with the sugar chain ratio in each sample.) It is a graph which shows the result of having quantified the abundance in each sample about the sugar_chain | carbohydrate determined with the significant difference by T-test performed with respect to 2 groups of a healthy person and esophageal cancer. (The total signal amount of sugar chain abundance was calculated and corrected with the sugar chain ratio in each sample.) It is a graph which shows the result of having quantified the abundance in each sample about the sugar_chain | carbohydrate determined to have a significant difference by T-test performed with respect to the group of healthy persons and stomach cancer. (The total signal amount of sugar chain abundance was calculated and corrected with the sugar chain ratio in each sample.) It is a figure which shows the result of having identified the peak combination which can be separated into 2 groups (a patient group and a healthy person group) by analyzing the area multivariately about each peak of a pancreatic cancer patient and a healthy person obtained as a result of mass spectrometry .

Hereinafter, the marker for diagnosis of digestive organ cancer and the method for examining digestive organ cancer of the present invention will be described in detail based on preferred embodiments.
<Gastrointestinal cancer marker>
A sugar chain is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and derivatives thereof are linked in a chain form by glycosidic bonds. .

  Sugar chains are very diverse and are substances that are involved in various functions of naturally occurring organisms. Sugar chains often exist as complex carbohydrates bound to proteins, lipids, and the like in vivo, and are one of the important components in vivo. It is becoming clear that sugar chains in living organisms are deeply involved in cell-to-cell information transmission, protein functions and interactions.

  Examples of biopolymers having sugar chains include proteoglycans on the cell wall of plant cells that contribute to cell stabilization, glycolipids that affect cell differentiation, proliferation, adhesion, migration, etc., and cell-cell interactions and cells. Glycoproteins involved in recognition can be mentioned, but the mechanism by which these high-molecular sugar chains control advanced and precise biological reactions while acting, assisting, amplifying, regulating, or inhibiting each other's functions gradually. It is being revealed. Furthermore, if the relationship between such sugar chains and cell differentiation / proliferation, cell adhesion, immunity, and cell carcinogenesis is clarified, this sugar chain engineering and medicine, cell engineering, or organ engineering are closely related. New developments are expected in relation to

  In particular, it has been revealed that sugar chains present on the cell surface play an important role as scaffolds for various biological reactions. For example, it is said to be involved in the occurrence of diseases due to abnormal interaction with the receptor, infection with AIDS virus, influenza virus, etc., and invasion of pathogenic E. coli O157 toxin and cholera toxin into cells.

  Furthermore, specific sugar chains appear on the cell surface in certain types of cancer cells, and the cell surface sugar chains are considered to be important molecules that give the cells individuality.

  Among the sugar chains (glycoproteins) as described above, the “N-linked sugar chain” is a sugar chain bonded to the nitrogen atom of the side chain amide group of the asparagine residue of the protein, and is N-type. It is also called a sugar chain or asparagine-linked sugar chain.

  The present inventor paid attention to such an N-linked sugar chain, in particular, an N-linked sugar chain contained in a blood sample of a digestive cancer patient. Tried development.

  Specifically, 17 patients with pancreatic cancer, 34 patients with esophageal cancer, and 22 patients with gastric cancer with consent were taken as subjects, blood was collected from each subject, and N-linked sugar chains in plasma were collected. Mass spectrometry was performed. As a result, the N-linked sugar chain (hereinafter simply referred to as “sugar chain”) in which the abundance is significantly changed in the blood sample of the subject (cancer patient) compared to the healthy person. ) Was successfully identified, and the present invention was completed.

  That is, the identified N-linked sugar chain is used as a digestive cancer diagnostic marker for identifying the presence or absence of gastrointestinal cancer, and is released from glycoprotein contained in blood. It is at least 1 sort (s) among what is represented by Formula (1)-following formula (6), It is characterized by the above-mentioned.

  Such sugar chains can be identified, for example, by mass spectrometry using a MALDI-TOF-MS type analyzer.

  Specifically, the N-linked sugar chain represented by the formula (1) has a peak mass-to-charge ratio (m / z) of 2521 when subjected to mass spectrometry using a MALDI-TOF-MS analyzer. It will be. The N-linked sugar chain represented by the formula (2) has a peak mass-to-charge ratio (m / z) of 2216, and the N-linked sugar chain represented by the formula (3) The mass-to-charge ratio (m / z) of the peak is 2216, and the N-linked sugar chain represented by the formula (4) has a mass-to-charge ratio (m / z) of 2054, and the formula (5) The N-linked sugar chain represented by the formula has a peak mass-to-charge ratio (m / z) of 2681, and the N-linked sugar chain represented by the formula (6) has a peak mass-to-charge ratio ( m / z) is 3108.

  In the mass spectrometry using the MALDI-TOF-MS type analyzer, a peak with a mass-to-charge ratio (m / z) of 2695 is recognized, and if an N-linked sugar chain showing this peak can be identified Similarly, this N-linked sugar chain can also be used as a marker for diagnosis of digestive organ cancer for identifying the presence or absence of digestive organ cancer.

“MALDI-TOF-MS” is a method of measuring mass based on time of flight using the MALDI method. Specifically, in the MALDI method, after spotting a sample on a plate, a matrix solution (2,5-dihydroxybenzoic acid) is added, dried and crystallized, and a large amount of energy is given to the matrix by pulse laser irradiation. , (M + H) ⁺ , (M + Na) ⁺ and other sample-derived ions and matrix-derived ions are desorbed. When an ion is accelerated at a constant acceleration voltage V, the ion mass is m, the ion velocity is v, the ion charge number is z, the elementary charge is e, and the ion flight time is t. The mass-to-charge ratio (m / z) can be expressed by “m / z = ² eVt ² / L ² ”.

  In the blood sample of the subject (cancer patient) as described above, a sugar chain (marker for digestive organ cancer diagnosis) whose abundance is significantly changed as compared with a healthy person is as follows. In addition, digestive organ cancer can be examined by the method for examining digestive organ cancer of the present invention.

<Testing method for digestive organ cancer>
The method for examining digestive organ cancer according to the present invention comprises a detection step of detecting an N-linked sugar chain represented by the formula (1) to the formula (6) released from a glycoprotein contained in blood; And an identification step for identifying the presence or absence of digestive organ cancer based on the detection result of the detection step.

  [1] First, N-linked sugar chains represented by the above formulas (1) to (6) that are released from glycoproteins contained in blood are detected (detection step).

[1-1] First, blood is collected from a subject as a sample.
The “blood collected from the subject” may be whole blood containing all blood components or serum or plasma separated from blood, but serum or plasma is good, especially plasma. Is preferred. Thereby, the N-linked sugar chain can be detected from the sample with excellent detection sensitivity.

[1-2] Next, sugar chains are released from glycoproteins in blood.
Such a release method is not particularly limited. For example, an enzyme method using N-glycosidase F (also referred to as glycopeptidase, PNGase, glycanase, glycoamidase, etc.) or glycopeptidase A, A hydrazine decomposition method can be mentioned. Among these, an enzyme method using N-glycosidase F can be preferably used. Thereby, among the sugar chains linked to the glycoprotein, N-linked sugar chains can be selectively released.

  When the enzyme method is used, a protease such as trypsin can be used in combination.

[1-3] Next, the sugar chain released in blood is purified. Such a purification method is not particularly limited as long as it is a method for selectively capturing and purifying sugar chains from a mixture in a sample, but MALDI- A method using BlotGlyco (registered trademark) (manufactured by Sumitomo Bakelite Co., Ltd.), which is a sugar chain-capturing bead (capture carrier) optimized for high-sensitivity measurement in TOF-MS or high performance liquid chromatography (HPLC) It is. Thereby, the sugar chain can be purified from the sample at an excellent purification rate.

  [1-4] Next, N-linked sugar chains represented by the above formulas (1) to (6) are detected from the purified sugar chains.

  In addition, the detection of such N-linked sugar chains represented by the formula (1) to the formula (6) is performed by mass spectrometry using a MALDI-TOF-MS type analyzer as described above. It can be performed with excellent accuracy.

  [2] Next, the presence or absence of the digestive organ cancer is identified based on the detection result of the detection step (identification step).

  Here, the mass-to-charge ratio (m / z) in the mass spectrometry of N-linked sugar chains in plasma in which 17 patients with pancreatic cancer, 34 patients with esophageal cancer, and 22 patients with gastric cancer were subject as described above. However, the detection value of the sugar chain indicating any of 2521, 2216, and 2054 was significantly lower than the detection value of the healthy subject.

  On the other hand, the detection value of the sugar chain whose mass-to-charge ratio (m / z) indicates either 2681 or 3108 was significantly higher than the detection value of the healthy subject.

  Therefore, in a subject, when the mass-to-charge ratio [m / z] has a detection value of at least one of 2521, 2216, and 2054 lower than that of a healthy person, the subject is treated with digestive cancer. Can be identified as suspected of being affected.

  In addition, if at least one detection value of the mass-to-charge ratio [m / z] of 2681 and 3108 is higher than that of a healthy person, the subject is suspected of suffering from digestive organ cancer. Can be identified.

  It should be noted that the detection value with a mass-to-charge ratio (m / z) 2695 in which the sugar chain structure is not specified was significantly higher than the detection value of a healthy person, and thus this detection value was normal. If it is higher than the person's detected value, the subject can be identified as suspected of having digestive cancer.

  As described above, a simple operation of detecting an N-linked sugar chain represented by the following formula (1) to the following formula (6) from the blood makes it easy to test for gastrointestinal cancer at an early stage. The treatment of digestive organ cancer can be performed at an early stage.

  In such a digestive cancer test method, it is possible to improve the accuracy by performing a digestive cancer test by combining detection results of a plurality of sugar chains among the sugar chains. Furthermore, it is possible to increase the accuracy of the examination for digestive organ cancer by combining the examination method for cancer in the present invention with other examination methods.

For example, the following results have been obtained by the study of the present inventor.
That is, the N-linked sugar chain released from the glycoprotein contained in the collected blood was divided into two groups of pancreatic cancer patients and healthy individuals, and peaks were identified using mass spectrometry. Then, peaks common to pancreatic cancer patients were collected, and the areas of each peak of pancreatic cancer patients and healthy subjects were subjected to multivariate analysis to obtain peak combinations that can be separated into two groups (patient group, healthy subject group). , Sugar chains of 1326, 1892, 2054, 2172, 2216, 2257, 2334, 2375, 2521, 2639, 2681, 2695, 2703, 2725, 2827, 3030 and 3108 are detected. The result is obtained.

  Therefore, in addition to sugar chains having a mass-to-charge ratio (m / z) of 2521, 2216, 2054, 2681, 3108 and 2695, the mass-to-charge ratio (m / z) of 1326, 1892, 2172, 2257, 2334, 2375 , 2639, 2703, 2725, 2827, and 3030, if the detected value of the sugar chain is deviated from the detected value of the healthy subject, this subject is identified as further suspected of having digestive organ cancer Thus, the accuracy of the examination for digestive organ cancer can be improved.

Note that a sugar chain having a mass-to-charge peak ratio (m / z) of 1326 by mass spectrometry using a MALDI-TOF-MS analyzer is 1326 as a result of mass analysis by a MALDI-TOF-MS analyzer, 1326 m. / Z is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₂ (Deoxyhexose) ₁ (Table 1).

The sugar chain having a peak mass-to-charge ratio (m / z) of 1892 by mass spectrometry using a MALDI-TOF-MS type analyzer is 1892 m / z as a result of mass analysis by a MALDI-TOF-MS type analyzer. It is a sugar chain that exhibits a mass spectrum peak, and its structural formula is presumed to be (HexNAc) ₂ (Deoxyhexose) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a mass-to-charge ratio (m / z) of a peak of 2172 by mass spectrometry using a MALDI-TOF-MS type analyzer is 2172 m / z as a result of mass analysis by a MALDI-TOF-MS type analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₁ (NeuAc) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a peak mass-to-charge ratio (m / z) of 2257 by mass analysis using a MALDI-TOF-MS type analyzer is 2257 m / z as a result of mass analysis by a MALDI-TOF-MS type analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₁ (HexNAc) ₃ (Deoxyhexose) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a peak mass-to-charge ratio (m / z) of 2334 by mass spectrometry using a MALDI-TOF-MS analyzer is 2334 m / z as a result of mass analysis by a MALDI-TOF-MS analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₃ (HexNAc) ₁ (NeuAc) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a peak mass-to-charge ratio (m / z) of 2375 by mass analysis using a MALDI-TOF-MS type analyzer is 2375 m / z as a result of mass analysis using a MALDI-TOF-MS type analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₂ (NeuAc) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a mass-to-charge peak ratio (m / z) of 2639 by mass analysis using a MALDI-TOF-MS type analyzer is 2639 m / z as a result of mass analysis by a MALDI-TOF-MS type analyzer. It is a sugar chain that exhibits a mass spectrum peak, and its structural formula is presumed to be (Hex) ₃ (HexNAc) ₄ + (Man) ₃ (GlcNAc) ₂ (Table 1).

  The sugar chain having a peak mass-to-charge ratio (m / z) of 2703 by mass spectrometry using a MALDI-TOF-MS type analyzer is 2703 m / z as a result of mass spectrometry using a MALDI-TOF-MS type analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structure is presumed to be the Na adduct of the sugar chain exhibiting the 2681 m / z mass spectrum peak (Table 1).

A sugar chain having a peak mass-to-charge ratio (m / z) of 2725 by mass spectrometry using a MALDI-TOF-MS type analyzer is 2725 m / z as a result of mass spectrometry using a MALDI-TOF-MS type analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₃ (Deoxyhexose) ₁ (NeuAc) ₁ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a mass-to-charge ratio (m / z) of 2827 by mass analysis using a MALDI-TOF-MS analyzer is 2827 m / z as a result of mass analysis by a MALDI-TOF-MS analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₂ (Deoxyhexose) ₁ (NeuAc) ₂ + (Man) ₃ (GlcNAc) ₂ (Table 1).

A sugar chain having a peak mass-to-charge ratio (m / z) of 3030 by mass spectrometry using a MALDI-TOF-MS analyzer is 3030 m / z as a result of mass analysis by a MALDI-TOF-MS analyzer. It is a sugar chain exhibiting a mass spectrum peak, and its structural formula is presumed to be (Hex) ₂ (HexNAc) ₃ (Deoxyhexose) ₁ (NeuAc) ₂ + (Man) ₃ (GlcNAc) ₂ (Table 1).

  The digestive cancer diagnostic marker and digestive cancer test method of the present invention have been described above based on the embodiments, but the present invention is not limited thereto.

  For example, an optional step may be added to the digestive cancer test method of the present invention as necessary.

  In addition, the “digestive system cancer” to which the digestive cancer test method of the present invention is applied is not particularly limited. However, the present invention is intractable pancreatic cancer, esophageal cancer, and occurrence frequency. It is particularly useful in gastric cancer, which is a high cancer.

  Furthermore, as long as the sugar chain is detected, an analyzer other than the MALDI-TOF-MS type analyzer can be used. As an ion source, for example, an electron ionization method, a chemical ionization method, an electric field detachment method, a fast atom collision method, an electrospray ionization method, an atmospheric pressure chemical ionization method, or the like can be used. A method such as a deflection type, a quadrupole type, an ion trap type, or a Fourier transform ion cyclotron resonance type can be used. In the present invention, as long as the sugar chain is detected, high performance liquid chromatography can be used, and the mass spectrometry and high performance liquid chromatography can be used in combination.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example.

[Example 1]
(1) Collection of blood Blood was collected from 17 patients with pancreatic cancer, 34 patients with esophageal cancer, 22 patients with gastric cancer, and 11 healthy persons as controls, with informed consent. Centrifuged. The obtained specimen (plasma) was anonymized for connection and then stored frozen at -80 ° C.

(2) Preparation of blood sample For the purpose of releasing proteins and modified sugar chains, plasma for each subject was treated with N-glycosidase F and trypsin. Specifically, pure water (165 μL), 1M ammonium bicarbonate (25 μL), and 120 mM dithiothreitol (25 μL) were added to 100 μL of plasma, allowed to stand at 60 ° C. for 30 minutes, and then 123 mM iodoacetamide (50 μL). ) And left at room temperature for 1 hour under light shielding. Subsequently, trypsin (2000 units, 25 μL) was added and allowed to stand at 37 ° C. for 1 hour, followed by heating at 80 ° C. for 15 minutes to denature trypsin. After cooling to room temperature, N-glycosidase F (10 units, 10 μL) was added and allowed to stand at 37 ° C. overnight. The enzyme was denatured by heating at 80 ° C. for 15 minutes to obtain a final amount of 400 μL of the enzyme-treated plasma sample.

  Further, an internal standard sugar chain solution was prepared by dissolving the internal standard glucose oligomer (1-20) (Seikagaku Corporation # 800111) in pure water so as to be 10 mg / mL.

  Subsequently, 5 μL (= 50 μg equivalent) of the internal standard sugar chain solution was added to 95 μL of the enzyme-treated plasma sample to prepare a total solution of 100 μL. Among these, 20 μL was treated with a sugar chain supplemented bead (BlotGlyco (registered trademark) for MALDI (manufactured by Sumitomo Bakelite Co., Ltd.)) to capture and label the released sugar chain.

(3) Analysis of results of sugar chain analysis The sugar chain captured by the sugar chain supplemented beads was purified and separated, mixed with a matrix (2,5-dihydroxybenzoic acid) solution, and subjected to MALDI-TOF-MS measurement. As a mass spectrometer, Autoflex III smartbeam TOF / TOF (manufactured by Bruker Daltonics) was used.

  In addition, software (for example, flexControl, flexAnalysis) attached to the device was used for data collection and analysis. Laser ionization was usually performed in positive ion mode and detected in reflector mode.

20 to 100 peaks were identified from the obtained mass spectrum.
Among these, for pancreatic cancer, gastric cancer, and pancreatic cancer, 6 types of sugar chains, excluding internal standard sugar chains, were analyzed, and each sugar chain was quantified by comparison with the internal standard. The total signal amount of the sugar chain abundance was calculated and corrected with the ratio of each sugar chain.

  Furthermore, T-test was performed between two groups of healthy subjects and pancreatic cancer, healthy subjects and esophageal cancer, and healthy subjects and gastric cancer, respectively, and an attempt was made to identify sugar chains showing significant differences.

  As a result, sugar chains having mass-to-charge ratios (m / z) of 2521, 2216, 2054, 2681, 3108, and 2695 are obtained for pancreatic cancer and esophageal cancer, and mass-to-charge ratios (m / z) are used for gastric cancer. ) Were identified as glycans showing 2521, 2216, and 2054 (see FIGS. 1-3).

  The peak area / total peak area (%) is significantly lower in cancer patients with sugar chains having a mass-to-charge ratio (m / z) of 2521, 2216, or 2054, indicating 2681, 3108, or 2695. Sugar chains were significantly higher in cancer patients.

  In addition, the correct answer rate when using 2521 m / z sugar chain and 2216 m / z sugar chain was calculated using six classifiers (Compound Covariate Predictor, Diagonal Linear Discriminant Analysis, 1-Nearest Neighbor Predictor, 3-Nearest Neighbor Predictor, Nearest Centroid Predictor and Support Vector Machine Predictor). As a result, a correct answer rate of about 88% on average was obtained for pancreatic cancer.

  In addition, the structural formula of the sugar chain was predicted from the signal obtained as a result of mass spectrometry. As a result, the sugar chain having a mass-to-charge ratio (m / z) of 2521 has the structure

The sugar chain that is expected to have a mass-to-charge ratio (m / z) of 2216 has the structure

Or

The sugar chain that is expected to have a mass-to-charge ratio (m / z) of 2054 has the structure

The sugar chain that is expected to have a mass-to-charge ratio (m / z) of 2681 has the structure

The sugar chain that is expected to have a mass-to-charge ratio (m / z) of 3108 has the structure

It was expected.
In addition, the structure of a sugar chain having a mass-to-charge ratio (m / z) of 2695 could not be predicted.

[Example 2]
(1) Collection of blood Blood was collected from 17 patients with pancreatic cancer with informed consent and 11 healthy subjects as controls, and the plasma was centrifuged. The obtained specimen (plasma) was anonymized for connection and then stored frozen at -80 ° C.

(2) Preparation of blood sample For the purpose of releasing proteins and modified sugar chains, plasma was treated with N-glycosidase F and trypsin. Specifically, pure water (165 μL), 1M ammonium bicarbonate (25 μL), and 120 mM dithiothreitol (25 μL) were added to 100 μL of plasma, allowed to stand at 60 ° C. for 30 minutes, and then 123 mM iodoacetamide (50 μL). ) And left at room temperature for 1 hour under light shielding. Subsequently, trypsin (2000 units, 25 μL) was added and allowed to stand at 37 ° C. for 1 hour, followed by heating at 80 ° C. for 15 minutes to denature trypsin. After cooling to room temperature, N-glycosidase F (10 units, 10 μL) was added and allowed to stand at 37 ° C. overnight. The enzyme was denatured by heating at 80 ° C. for 15 minutes to obtain a final amount of 400 μL of the enzyme-treated plasma sample.

  Further, an internal standard sugar chain solution was prepared by dissolving the internal standard glucose oligomer (1-20) (Seikagaku Corporation # 800111) in pure water so as to be 10 mg / mL.

  Subsequently, 5 μL (= 50 μg equivalent) of the internal standard sugar chain solution was added to 95 μL of the enzyme-treated plasma sample to prepare a total solution of 100 μL. Among these, 20 μL was treated with a sugar chain supplemented bead (BlotGlyco (registered trademark) for MALDI (manufactured by Sumitomo Bakelite Co., Ltd.)) to capture and label the released sugar chain.

(3) Analysis of results of sugar chain analysis The sugar chain captured by the sugar chain supplemented beads was purified and separated, mixed with a matrix (2,5-dihydroxybenzoic acid) solution, and subjected to MALDI-TOF-MS measurement. As a mass spectrometer, Autoflex III smartbeam TOF / TOF (manufactured by Bruker Daltonics) was used.

  For data collection and analysis, software attached to the device (for example, flexControl, flexAnalysis) was used. Laser ionization was usually performed in positive ion mode and detected in reflector mode.

  20 to 100 peaks were identified from the obtained mass spectrum. Peaks common to pancreatic cancer patients are collected, and the areas of each peak of pancreatic cancer patients and healthy subjects are analyzed using multivariate analysis software SIMCA-P / P + (manufactured by UMETRICS), and two groups (patient group, healthy) The peak combinations that can be separated into a group of individuals were determined.

  As a result, as sugar chain peaks giving the above two-group separation, mass to charge ratios (m / z) are 1326, 1892, 2054, 2172, 2216, 2257, 2334, 2375, 2521, 2639, 2681, 2703, 2725, Sugar chains 2827, 3030 and 3108 were detected (FIG. 4, Table 1).

In addition, the meaning of the symbol in Table 1 is as follows.
δmass = [measured value m / z]-[theoretical value m / z]
+: The structure shown on the right side of “+” is a basic structure, and the structure shown on the left side is an additional structure.

Hex: hexose (mannose)
HexNAc: N-acetylhexosamine (N-acetylglucosamine)
Deoxyhexose: fucose
Man: mannose
GlcNAc: N-acetylglycosamine
NeuAc: N-acetylneuraminic acid
It should be understood that the values of the mass-to-charge ratio (m / z) shown in FIGS.

Claims (12)

Used to identify the presence or absence of gastrointestinal cancer,
N-linked sugar chains released from glycoproteins contained in blood,
The N-linked sugar chain is at least one of those represented by the following formula (1) to the following formula (6).

  The digestive apparatus according to claim 1, wherein the N-linked sugar chain represented by the formula (1) has a mass-to-charge ratio of 2521 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The digestive organ according to claim 1, wherein the N-linked sugar chain represented by the formula (2) has a mass-to-charge ratio of 2216 m / z as measured by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The digestive organ according to claim 1, wherein the N-linked sugar chain represented by the formula (3) has a mass-to-charge ratio of 2216 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The digestive apparatus according to claim 1, wherein the N-linked sugar chain represented by the formula (4) has a mass-to-charge ratio of 2054 m / z as measured by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The digestive apparatus according to claim 1, wherein the N-linked sugar chain represented by the formula (5) has a mass-to-charge ratio of 2681 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The digestive organ according to claim 1, wherein the N-linked sugar chain represented by the formula (6) has a mass-to-charge ratio of 3108 m / z by mass spectrometry using a MALDI-TOF-MS type analyzer. Cancer diagnostic marker.   The marker for digestive organ cancer diagnosis according to any one of claims 1 to 7, wherein the digestive organ cancer is pancreatic cancer, esophageal cancer, or stomach cancer. A test method for digestive cancer that identifies the presence or absence of digestive cancer using the marker for diagnosis of digestive cancer according to claim 1,
A detection step of detecting an N-linked sugar chain represented by the formula (1) to the formula (6) released from the glycoprotein;
A method for examining digestive organ cancer, comprising: an identification step for identifying the presence or absence of the digestive organ cancer based on the detection result of the detection step.   The digestion according to claim 9, wherein in the detection step, detection of the N-linked sugar chain represented by the formula (1) to the formula (6) is performed by mass spectrometry using a MALDI-TOF-MS analyzer. Test method for organ cancer.   The mass-to-charge ratio [m / z] is identified as suspected of having gastrointestinal cancer if the detection value of at least one of 2521, 2216 and 2054 is lower than that of a healthy person. The test | inspection method of digestive organ cancer of description.   The mass-to-charge ratio [m / z] is identified as suspected of having gastrointestinal cancer when the detection value of at least one of 2681 and 3108 is higher than that of a healthy person. Of digestive system cancer.

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